The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to optical probes for post translational type modification activities, such as phosphorylation, and methods for their use.
Systems and methods for rapidly identifying chemicals with biological activity in samples, especially small liquid samples, is of particular relevance to the agrochemical and pharmaceutical fields. Various strategies are typically used to reduce processing times and associated costs of screening large numbers of chemical entities, including simplified assay design, automation, robotics and miniaturization of sample size. The advent of high throughput analysis and increasing use of miniaturized formats has led to the development of high density plate formats. For example, containing 384, 864 and 3456 wells as described in U.S. patent application Ser. No. 08/868,049 Entitled xe2x80x9cLow Background Multi-Well Plates with greater than 864 Wells for Fluorescence Measurements of Biological and Biochemical Samples,xe2x80x9d filed Jul. 3, 1997, now pending. Even higher density sample processing systems, for example using chips that contain miniaturized microfluidic devices are being developed (see, for example, R and D Magazine, November 1998, pages 38 to 43 entitled xe2x80x9cLab-on-a Chip: Biotech""s next California Gold Rushxe2x80x9d).
Higher density plates enable faster analysis and handling of large sample or chemical libraries, such as in automated screening systems, but place considerable constraints on the assays that can be successfully employed within them. In particular, there is a need to develop assays that are compatible with miniaturized systems and which give accurate and reproducible assay results. Central to this need is a requirement for high sensitivity assays based on optical analysis, such as fluorescence or luminescence that do not require wash steps (e.g. xe2x80x9caddition only assaysxe2x80x9d).
One of the largest and most important classes of intracellular activities for which drugs may be particularly valuable are those involved in post-translational modification activities. These activities are typically directed to the modification of proteins and nucleic acids within living cells to effect changes in the biological activity and function of these molecules. The major methods of protein or polypeptide, post-translational modification include protein phosphorylation, methylation, prenylation, glycosylation, ubiquitination sulfation and proteolysis (see generally Cells. A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998) review). Major methods of nucleic acid modification include methylation, ADP-ribsoylation and restriction digestion. A variety of environmental stimuli such as the presence of growth factors, hormones, changes in the cell cycle and toxins can transiently modulate the post-translational state of many intracellular components. The rapid development of specific, and effective inhibitors for a particular post-translational activity requires the development of suitable assays that can reliably and -sensitively detect these activities in a high throughput screening system.
In spite of their great potential importance however, there are few existing methods of measuring such activities that are homogenous, non radioactive and sensitive enough to accurately and reproducible work in high throughput, or ultra high throughput screening systems. Such assays, by reducing the time required to identify and develop useful chemicals, can dramatically increase the value of a new drug by enabling its patentability and increasing it""s period of exclusivity in the market.
Examples of such post-translational activities include, amongst others, protein methylation and prenylation. Protein prenylation involves the addition of isoprenoid moieties such as farnesyl and geranylgeranyl to proteins, and is a major mechanism of post-translational modification for many membrane-associated proteins. (Clark, 1992 Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu. Rev. Biochem. 61 355-386). In most cases, the amino acid derivatized with the isoprenoid is a cysteine, or cysteines close to the carboxyl-terminus of the protein. Present methods of measuring protein prenylation and methylation typically involve labeling cells with radioactive precursors such as [3H]-mevalonate or [3H]-S-adenosylmethionine, isolation of the protein of interest and measurement of radioactive incorporation. There is thus a need for assays for these activities that are sensitive, simple to use, non-radioactive and adaptable to high throughput screening methods.
Another important example of post-translational modification is protein glycosylation, which plays an extremely important role in the function of a significant number of proteins (Varki, 1993, Biological roles of oligosaccharides. Glycobiology 3 97-130). Protein glycosylation, unlike most other types of post-translational modification provides a wide diversity in the oligosaccharides added to a protein because of the potential for branching after the addition of the first sugar residue. Present methods of measuring glycosylation typically involve determining radioactive incorporation of a precursor oligosaccharide into a protein, isolating the protein and then measuring specific radioactive incorporation into a protein. There is thus a need for fluorescence or luminescence based assays for these activities that are adaptable to high throughput screening methods. It is one objective of the present invention to provide optical probes and methods of use that meet this need.
Protein kinases and phosphatases are generally recognized as one of the more important general mechanisms of regulating protein function. A recent review and analysis of diseases associated with genetic defects in protein kinases lists over 400 specific disease states associated with these activities alone. Protein kinases act on proteins via the addition of phosphate groups (phosphorylation) primarily on the amino acids, tyrosine, serine or threonine. Protein phosphatases in contrast, act to remove these phosphate groups thereby reversing the effects of phosphorylation. Changes in the phosphorylation state of proteins, can regulate the enzymatic activity, protein localization and protein-protein interactions of a particular protein within a cell. Such changes can subsequently modulate virtually every aspect of cellular metabolism, regulation, growth and differentiation. The overall balance of kinase and phosphatase activities in a cell is a primary determinant of the phosphorylation state of a protein at any one time.
However, current methods of measuring protein kinases, have many disadvantages, which prevents or hampers the ability to rapidly screen for drugs using miniaturized automated formats of many thousands of compounds.
For example, many current methods of measuring their activity rely on the incorporation and measurement of 32P into the protein substrates of interest. In whole cells, this necessitates the use of high levels of radioactivity to efficiently label the cellular ATP pool and to ensure that the target protein is efficiently labeled with radioactivity. After incubation with test drugs, the cells must be lysed and the protein of interest purified to determine its relative degree of phosphorylation. This method requires large numbers of cells, long preincubation times, careful manipulation, and washing steps to avoid artifactual phosphorylation or dephosphorylation. Furthermore, this kinase assay approach requires purification of the target protein, and final radioactive incorporation into target proteins is usually very low giving the assay poor sensitivity. In high throughput screening operations, this approach requires large amounts of radioactivity, which can be an environmental and health hazard.
Alternative kinase assay methods, such as those based on phosphorylation-specific antibodies using ELISA-type approaches, involve the difficulty of producing antibodies that distinguish between phosphorylated and non-phosphorylated proteins.
Furthermore, most kinase measurements have the requirement for cell lysis, multiple incubations, and washing stages that are time consuming, complex to automate, and potentially susceptible to artifacts.
There is thus a need for assays for enzymes, such as those involved in post-translational modification, that are sensitive, simple to use, applicable to virtually any activity and adaptable to high throughput screening methods. Preferably, such assays would not utilize radioactive materials so that the assays would be safe and not generate hazardous wastes. The present invention addresses these needs, and provides additional benefits as well.
This invention provides a fluorescent or bioluminescent substrate useful as an optical probe or sensor of post translational type modifications, such as phosphorylation. In one embodiment, the invention comprises a polypeptide moiety, which contains a recognition motif for a post translational type activity and a protease site, which is coupled to a probe moiety. Typically, the presence of a modification at the recognition motif alters protease activity at the protease site resulting in a modulation of the cleavage rate of the protease. Cleavage is sensed by a measurable change in at least one optical property of the optical probe upon cleavage at the protease site, FIG. 1.
In one embodiment the probe is a fluorescent or luminescent moiety.
In another embodiment, the invention further comprises a fluorescent quencher coupled to the polypeptide that quenches emission from the first probe moiety. In this embodiment, the first probe moiety and the quencher moiety are coupled to the polypeptide such that the recognition motif and the protease site are located between them (FIG. 1). In this case, cleavage of the polypeptide by a protease results in an alteration in the fluorescence emission of the first probe moiety that may be used to determine post-translational activity.
In another embodiment, the optical probe may further comprise a second probe moiety coupled to the polypeptide that participates in energy transfer with the first probe moiety. In this embodiment, the first probe moiety and the second probe moiety are coupled to the polypeptide such that recognition motif and the protease site are located between them. In this case, cleavage of the polypeptide by a protease results in an alteration in energy transfer between the first probe moiety and the second probe moiety that may be used to determine post-translational activity.
The invention also provides methods for using the optical probes of the invention to determine whether a sample contains a post-translational type modification activity such as protein phosphorylation or dephosphorylation, methylation, prenylation or glycosylation. The method consisting of; i), contacting the optical probe with a sample, usually containing or suspected of containing a post translational type activity; ii), contacting the sample and optical probe with a protease, and iii), determining at least one optical property of said optical probe, or product thereof.
In another embodiment, the invention provides methods for using the optical probes of the invention to determine whether a test chemical modulates the activity of a post-translational type activity.
In another aspect, the invention provides a library of optical probes, each with a unique peptide sequence for use in selecting an optimal sequence specificity of a post-translational type activity.
Another aspect of the present invention includes a compound or therapeutic identified by at least one method of the present invention. These methods can include monitoring the efficacy and/or toxicology of said therapeutic in an in vitro or in vivo model. The compound can be provided in therapeutically acceptable carrier and can form a therapeutic composition.
A further aspect of the present invention includes various systems for spectroscopic measurements. In one embodiment, the system typically includes at least one reagent for an assay and a device, said device comprising a container and a platform. The container can include the optical sensor compounds of the present invention, and additional reagents necessary for the post-translational type activity. Addition of a sample to the container, followed by the addition of a protease after a given time results in a change in at least one fluorescent property of the optical probes of the present invention that can be used to determine the post-translational type activity of the sample.
In another embodiment the system can include a microfluidic spectroscopic system comprising at least one fluid containing structure with at least one electro-osmotic or electrophoretic system to control fluid movement within that structure.